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Blood, Vol. 93 No. 7 (April 1), 1999:
pp. 2386-2394
Sodium Salicylate Activates Caspases and Induces Apoptosis of Myeloid
Leukemia Cell Lines
By
Lidija Klampfer,
Jörg Cammenga,
Hans-Georg Wisniewski, and
Stephen D. Nimer
From the Laboratory of Molecular Aspects of Hematopoiesis and
Department of Medicine, Memorial Sloan-Kettering Cancer Center, New
York; and New York University Medical Center, Department of
Microbiology, New York, NY.
 |
ABSTRACT |
Nonsteroidal antiinflammatory agents (NSAIA) have been shown to
exert potent chemopreventive activity against colon, lung, and breast
cancers. In this study, we show that at pharmacological concentrations
(1 to 3 mmol/L) sodium salicylate (Na-Sal) can potently induce
programmed cell death in several human myeloid leukemia cell lines,
including TF-1, U937, CMK-1, HL-60, and Mo7e. TF-1 cells
undergo rapid apoptosis on treatment with Na-Sal, as indicated by
increased annexin V binding capacity, cpp-32 (caspase-3) activation,
and cleavage of poly (ADP-ribose) polymerase (PARP) and
gelsolin. In addition, the expression of MCL-1, an antiapoptotic member
of the BCL-2 family, is downregulated during Na-Sal-induced cell
death, whereas the expression of BCL-2, BAX, and BCL-XL is unchanged. Z-VAD, a potent caspase inhibitor, prevents the cleavage of
PARP and gelsolin and rescues cells from Na-Sal-induced apoptosis. In
addition, we show that Na-Sal accelerates growth factor
withdrawal-induced apoptosis and synergizes with daunorubicin to induce
apoptosis in TF-1 cells. Thus, our data provide a potential mechanism
for the chemopreventive activity of NSAIA and suggest that salicylates may have therapeutic potential for the treatment of human leukemia.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
NONSTEROIDAL antiinflammatory agents
(NSAIA) are known for their potent analgesic, antipyretic, and
antiinflammatory activity, which is largely ascribed to their
well-documented ability to inhibit prostaglandin synthesis. In
addition, NSAIA have potent chemopreventive activity.1-4
Inhibition of COX2 has been suggested to mediate the chemopreventive
activity of NSAIA. However, much higher doses of NSAIA are required to
achieve an anticancer effect than to inhibit
cyclooxygenases,5 suggesting that the chemopreventive activity of NSAIA is not mediated solely through inhibition of prostaglandin synthesis. The chemopreventive activity of NSAIAs has
therefore been ascribed to their growth-inhibitory
properties,6 their ability to induce
differentiation,7 and to their ability to induce
apoptosis.8
Apoptosis (programmed cell death) ensures the homeostasis of tissues
during development, host defense, and aging. Aberrant cell survival due
to insufficient apoptosis has been linked to the development
and/or progression of human malignancies. In addition, cancer
chemotherapy agents act primarily through induction of apoptosis in
target cells, often in a p53-dependent manner.9-11 Accordingly, cancer cells with mutations in the p53 gene, or
abnormalities in the expression of other genes that regulate apoptosis,
can display intrinsic resistance to chemotherapy-induced apoptosis. This suggests that acquired defects in the apoptotic process play an
important role in the development of drug resistance.
The molecular mechanisms involved in salicylate-induced apoptosis are
largely unknown. Salicylates do interfere with mitogen-activated protein kinase (MAPK) signaling pathways; they have
been shown to inhibit tumor necrosis factor (TNF)-induced extracellular
signal-regulated kinase (ERK) and c-jun NH2
terminal protein kinase (JNK)/stress-activated protein kinase
(SAPK) activation,12,13 and to
activate12 or inhibit ultraviolet B-induced
p38 kinase.14 Activation of both JNK and p38 kinase, with
concurrent inhibition of ERK, has been shown to be critical for growth
factor withdrawal-induced apoptosis of PC-12 cells.15 In
addition, several transcription factors have been shown to be targets
of salicylate action, which may mediate salicylate-induced programmed
cell death. For example, salicylates are potent inhibitors of NF- B
activation,16 and inhibition of NF-B activity has been
linked to the induction of apoptosis.17-20 Salicylates also
inhibit AP-1 activity, either directly14,21 or through
p38-mediated inhibition of ATF-2 activity.22
Given the significant chemopreventive activity of NSAIA in colon
cancer, we examined whether sodium salicylate (Na-Sal) affects the
survival of human myeloid leukemia cells and investigated the molecular
mechanism of Na-Sal-induced apoptosis. We show that Na-Sal induces
rapid apoptosis of several human myeloid leukemia cell lines via
activation of caspase-3, subsequent cleavage of poly (ADP-ribose)
polymerase (PARP) and gelsolin, and inhibition of MCL-1 expression. The
central role of caspase activation in Na-Sal-induced apoptosis was
established by using the caspase inhibitor Z-VAD, which completely
prevented salicylate-induced cell death. In addition, we show that at
sublethal concentrations, Na-Sal accelerates apoptosis induced by
growth factor withdrawal or treatment with daunorubicin, an agent most
commonly used in the treatment of human leukemia. Unlike
acetyl-salicylic acid (aspirin), Na-Sal does not inhibit platelet
aggregation, thus the ability of Na-Sal to synergize with daunorubicin
in inducing apoptosis in myeloid leukemia cells suggests that Na-Sal
could potentially improve the treatment of human myeloid leukemia.
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MATERIALS AND METHODS |
Cell cultures.
TF-1 cells (kindly provided by Toshio Kitamura, University of Tokyo,
Tokyo, Japan) were grown in RPMI 1640 medium,
supplemented with 10% fetal bovine serum (FBS), 1% glutamine,
penicillin, streptomycin, and recombinant human granulocyte-macrophage
colony-stimulating factor (rhGM-CSF) (10 ng/mL) (kindly provided by
Novartis Corp, East Hanover, NJ).
3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) assays to determine the viability of TF-1 cells were performed according to the manufacturer's instructions
(Boehringer Mannheim, Indianapolis, IN).
Annexin V binding assay.
Flow cytometric analysis of annexin V-fluorescein isothiocyanate (FITC)
and propidium iodide (PI)-stained cells was performed using the
Apoptosis Detection Kit (R & D Systems, Minneapolis, MN) as recommended by the manufacturer. Briefly, cells
were washed with phosphate-buffered saline (PBS) and resuspended in 1x
binding buffer. A total of 10 µL of fluorescein-conjugated annexin V
and 10 µL of PI was added to the cells for 15 minutes at room
temperature. After incubation, 400 µL of 1× binding buffer was
added and cells were analyzed for annexin V binding within 1 hour using
flow cytometry.
Western blot analysis.
TF-1 cells were treated with different concentrations of Na-Sal for the
time indicated. Cell pellets were resuspended in sodium dodecyl sulfate
(SDS)-lysis buffer, and the samples were electrophoretically separated
on a 12% SDS-polyacrylamide gel electrophoresis (PAGE) gel. After
transfer to a nitrocellulose membrane, the gel was stained with
Coomassie blue to demonstrate equal loading of protein in all lanes.
The presence of BCL-2 protein was detected using a monoclonal antibody
that specifically recognizes BCL-2 (Oncogene Science, Cambridge, MA).
The membrane was reprobed with a polyclonal anti-MCL-1 antibody
(Pharmingen, San Diego, CA), an anti-Bax antibody (kindly provided by
John Reed, Burnham Institute, La Jolla, CA), an
anti-BCL-X antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The
anti-gelsolin antibody was purchased from Sigma (St Louis, MO), the caspase-3 antibody from Transduction
Laboratories (Lexington, KY), and the anti-PARP antibody from
Pharmingen. Western blots were developed using the
Amersham ECL kit (Amersham, Arlington Heights, IL) and
exposed to Kodak XAR film (Eastman Kodak, Rochester, NY).
Analysis of caspase-3 activity.
TF-1 cells were treated with increasing concentrations of Na-Sal, and
the enzymatic activity of caspase-3 was determined using ApoAlert
cpp32/caspase-3 assay kit (Clontech, Palo Alto, CA) as suggested by the
manufacturer. Briefly, 2 × 106 cells were resuspended
in 50 µL of lysis buffer and reaction buffer and a chromogenic cpp32
substrate DEVD-p-nitroanilide (DEVD-pNA) was added.
Reactions were incubated at 37°C for 1 hour and
samples were measured at 400 nm.
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RESULTS |
Na-Sal induces apoptosis of several hematopoietic cell lines.
We examined the effect of Na-Sal on the survival of TF-1 cells, a
GM-CSF- or interleukin-3 (IL-3)-dependent human myeloid leukemia cell
line. In contrast to primary fibroblasts, which undergo apoptosis after
prolonged treatment with relatively high concentrations of Na-Sal (20 mmol/L),12 TF-1 cells show clear signs of apoptosis on
treatment with as little as 4 mmol/L Na-Sal (Fig 1A). Na-Sal-treated TF-1 cells
display morphological signs of apoptosis including cell shrinkage,
membrane blebbing, and eventual disintegration into numerous apoptotic
bodies as soon as 5 hours after treatment (data not shown). TF-1 cells
cultured in the presence of 10 mmol/L Na-Sal for 10 hours showed less
than 10% viability (data not shown). We defined the sensitivity of several other myeloid leukemia cell lines to Na-Sal-induced apoptosis. CMK-1 and U937 cells were as sensitive to Na-Sal as TF-1 cells, whereas
K562 cells were less sensitive; some K562 cells survived despite
exposure to 20 mmol/L Na-Sal for 40 hours (Fig 1A). Both HL-60 and Mo7e
cells undergo apoptosis on treatment with 1 to 5 mmol/L Na-Sal (data
not shown).
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| Fig 1.
Na-Sal induces apoptosis of acute myeloid leukemia cell
lines. TF-1, U937, CMK-1, and K562 cells were either left untreated
( ) or were treated with 0.5 ( ), 1 ( ), 5 ( ), or 20 ( )
mmol/L Na-Sal for the time indicated. Viability was determined by the
trypan blue exclusion test. (B) Increased annexin V binding to TF-1
cells cultured in the absence (control) or in the presence of 4 mmol/L
Na-Sal for 1 hour, 4 hours, 10 hours, or 20 hours. Annexin V binding
and PI staining were analyzed by flow cytometry, as described in
Materials and Methods. The percentage of cells positive for annexin V
and/or PI is depicted in each quadrant.
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To study the molecular mechanism underlying Na-Sal-induced cell death,
we focused our studies on TF-1 cells, because we and others have
previously characterized the process of growth factor withdrawal-induced cell death in these cells23 (and
Klampfer et al, submitted). To confirm that the cell
death induced by Na-Sal was due to apoptosis, we stained TF-1 cells
with annexin V, which binds phosphatidylserine with high affinity.
Translocation of phosphatidylserine from the inner face of the plasma
membrane to the cell surface is one of the earliest events in apoptosis and can be analyzed by staining with FITC-labeled annexin
V.24 Cells were simultaneously stained with PI and analyzed
by flow cytometry (fluorescence-activated cell sorting [FACS]). We
observed a time-dependent increase in annexin V binding in
Na-Sal-treated cells (Fig 1B). Untreated TF-1 cells exerted low
background staining with annexin V (4%), but after incubation with 4 mmol/L Na-Sal for 4 hours, 12 % of cells stained positive with annexin
V and negative with PI, indicating that they were undergoing apoptosis. After 10 hours of treatment, 21% of the cells were annexin V positive and PI negative (Fig 1B). At this time point, an additional 16% of the
cells stained positive for both annexin V and PI, representing cells
undergoing late apoptosis/secondary necrosis, and 44% of cells stained
positive for PI after 20 hours of Na-Sal treatment.
Na-Sal inhibits the expression of MCL-1, but does not affect the
expression of BCL-2, BCL-XL, and Bax or activation of MAPK.
To elucidate the molecular basis for Na-Sal-induced apoptosis of TF-1
cells, we first examined whether Na-Sal interferes with GM-CSF
signaling. GM-CSF provides survival and proliferative signals via
activation of MAPK,25 whereas Na-Sal has been shown to
potently inhibit TNF-induced MAPK activation in human FS-4
fibroblasts.13 To determine if Na-Sal interferes with
GM-CSF-induced MAPK activation in TF-1 cells, we deprived cells of
GM-CSF for 16 hours and then incubated the cells with Na-Sal for 10 or
60 minutes before stimulation with GM-CSF for 10 minutes. Whole cell
lysates were examined by Western blotting for the presence of
phosphorylated MAPK. As shown in Fig 2A,
Na-Sal did not interfere with the phosphorylation of p42 or p44 after
treatment with GM-CSF, excluding the possibility that Na-Sal induces
cell death in TF-1 cells by interfering with GM-CSF-induced MAPK
activation.
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| Fig 2.
Na-Sal regulates the expression of MCL-1. (A) Na-Sal does
not effect GM-CSF-induced tyrosine phosphorylation of p42/p44 MAPK.
TF-1 cells were deprived of GM-CSF for 12 hours and were either left
untreated (lane 1) or stimulated with GM-CSF for 10 minutes (lanes 2 through 4). Some cells were preincubated with 5 mmol/L Na-Sal for 10 minutes (lane 3) or 60 minutes (lane 4) before being stimulated with
GM-CSF. Cell lysates were analyzed with a phospho-specific MAPK
antibody (New England Biolabs, Beverly, MA). (B) The expression of
BCL-2, Bax, and BCL-XL is not altered by Na-Sal treatment.
TF-1 cells were treated with increasing concentrations of Na-Sal (0.5, 1, 5, and 10 mmol/L) for 5 hours and the presence of BCL-2,
BCL-XL, and Bax was analyzed by Western blotting. (C)
Time-dependent and (D) dose-dependent inhibition of MCL-1 expression
after treatment with Na-Sal. Cells were treated with 5 mmol/L Na-Sal
for the indicated times (C) or were treated with the indicated
concentrations of Na-Sal for 5 hours (D). The level of MCL-1 protein
was determined by Western blotting. (E and F) Densitometric scanning of
(C) and (D), respectively. Scanning was performed using the Ambis
nonradioactive imaging system (Ambis, Inc, San Diego, CA).
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Next, we examined whether Na-Sal induces cell death by modulating the
expression of BCL-2 family members, which ultimately determine the
cell's response to apoptotic stimuli.26 Treatment of TF-1
cells with concentrations of Na-Sal that are sufficient to induce
apoptosis did not significantly alter the expression of the BCL-2,
BCL-XL, or Bax proteins after 5 hours (Fig 2B), or after 18 hours (data not shown). In contrast, the expression of MCL-1 protein
decreased in a dose- and time-dependent manner in salicylate-treated
cells (Fig 2C through F). Na-Sal treatment also decreased the level of
MCL-1 mRNA (data not shown), suggesting that downregulation of the
MCL-1 protein is the result of an effect on transcription or mRNA
stability. We and others have shown that MCL-1 plays an important role
in the GM-CSF-dependent survival of TF-1 cells,23
suggesting that downregulation of MCL-1 may also be important in the
Na-Sal-induced cell death of TF-1 cells.
Na-Sal activates cpp32 (caspase-3), and induces cleavage of PARP and
gelsolin.
A family of aspartate-specific cysteinyl proteases (caspases) play a
pivotal role in the execution of programmed cell
death,27,28 thus we examined whether treatment of cells
with Na-Sal results in activation of these enzymes.
Cpp32/Yama/apopain/caspase-3 has been shown to play an important role
in chemotherapy-induced,29 growth factor
withdrawal-induced,30 Fas-mediated,31 and
retinoic acid-induced apoptosis.32 To determine whether
activation of caspase-3 plays a role in Na-Sal-induced apoptosis, TF-1
cells were incubated with 5 mmol/L Na-Sal and analyzed by Western
blotting for proteolytic processing of caspase-3. Incubation of TF-1
cells with Na-Sal resulted in activation of caspase-3, as is evident by
the appearance of the 20-kD proteolytic product of cpp32 after 6 hours
of Na-Sal treatment (Fig 3A). Accordingly,
caspase-3 protease activity was increased in Na-Sal-treated cells as
measured by the colorimetric assay, using DEVD-pNA as a chromogenic
substrate (Fig 3B). The significant increase in caspase-3 activity at 5 mmol/L Na-Sal correlates well with the induction of apoptosis seen at
this concentration.
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| Fig 3.
Na-Sal treatment of TF-1 cells activates caspase-3 and
induces cleavage of PARP and gelsolin. (A and B) Activation of cpp-32
(caspase-3) in response to treatment with Na-Sal. (A) TF-1 cells were
treated with 5 mmol/L Na-Sal for the time indicated and the presence of
intact cpp-32 and its proteolytic fragment (p20) was shown by
immunoblotting using an anti-cpp-32 antibody (Transduction
Laboratories) (B) Caspase-3 activity was determined in cells treated
with Na-Sal (1 mmol/L, 3 mmol/L, and 5 mmol/L) for 16 hours. (C and D)
Na-Sal-induced cleavage of PARP. TF-1 cells were left untreated (0) or
were treated with 5 mmol/L Na-Sal for 2, 6, or 9 hours, as indicated
(C). Cells were also treated with increasing concentrations of Na-Sal
(1, 5, or 10 mmol/L, as indicated) for 5 hours (D). The presence of
PARP (116 kD) and its 86-kD proteolytic fragment were detected by
immunoblotting using an anti-PARP antibody (Pharmingen). (E)
Na-Sal-induced cleavage of gelsolin. TF-1 cells were treated with
increasing concentrations of Na-Sal (indicated at the top) for 5 hours;
the amount of gelsolin and its proteolytic degradation was analyzed by
immunoblotting using an anti-gelsolin antibody (Sigma).
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Activation of caspases leads to cell demise27,28 via
cleavage of cellular substrates, such as actin,33
fodrin,34 PARP,35 and gelsolin.36
To determine whether PARP is cleaved in Na-Sal-treated cells, we
treated cells with increasing concentrations of Na-Sal (1, 5, and 10 mmol/L) for 5 hours. As shown in Fig 3D, treatment of TF-1 cells with
Na-Sal results in a dose-dependent cleavage of PARP. TF-1 cells were
next treated with 5 mmol/L Na-Sal for increasing amounts of time (2, 6, or 9 hours). Cleavage of PARP was observed as soon as 2 hours after
treatment (Fig 3C), although a caspase-3 cleavage product was not
detected at that time point (Fig 3A). It is possible that the amount of
caspase-3 cleavage was too low to be detected by Western blotting or,
alternatively, that other caspases participate in Na-Sal-induced
cleavage of PARP.
Recent studies have shown that gelsolin is a substrate for caspase-3
and that expression of the 40-kD cleavage product of gelsolin induces
morphological features of apoptosis, such as cell rounding and the
formation of apoptotic bodies.36 To examine if gelsolin is
cleaved in Na-Sal-treated cells, we incubated TF-1 cells in the
presence of 1, 5, 10, or 20 mmol/L Na-Sal for 5 hours and examined cell
lysates for the presence of gelsolin and its cleavage product by
Western blotting. Treatment of cells with 5 mmol/L Na-Sal induces
cleavage of gelsolin, as seen by the appearance of its 40-kD cleavage
product (Fig 3E).
Z-VAD inhibits Na-Sal-induced caspase-3 activation, cleavage of PARP
and gelsolin, and apoptosis of TF-1 cells.
To evaluate the role of caspase-3 activation in Na-Sal-induced
apoptosis, we treated TF-1 cells with Na-Sal in the presence of a
caspase inhibitor. Z-VAD.fmk is a cell-permeable protease inhibitor,
which inhibits proteolytic processing of caspase-3 and prevents the
IL-3 withdrawal-induced apoptosis of 32D cells.31 TF-1
cells were treated with 5 mmol/L Na-Sal alone, or in the presence of
various concentrations of Z-VAD (10 µmol/L, 50 µmol/L, or 100 µmol/L). Whereas treatment with 10 µmol/L or 50 µmol/L Z-VAD had
little or no effect on Na-Sal-induced apoptosis (data not shown),
treatment of cells with 100 µmol/L Z-VAD inhibited Na-Sal-induced
apoptosis completely (Fig 4A).
Pretreatment of TF-1 cells with 100 µmol/L Z-VAD for 1 hour also
prevented the activation of caspase-3 protease activity (Fig
4B) and the cleavage of gelsolin and PARP in Na-Sal-treated cells
(Fig 4C and D). Similar results were obtained when cells were treated
with Z-VAD and Na-Sal simultaneously (data not shown). These results
confirm the key role of caspase activation and subsequent cleavage of
PARP and gelsolin in Na-Sal-induced apoptosis.
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| Fig 4.
z-VAD prevents Na-Sal-induced apoptosis of TF-1 cells.
(A) Morphological changes. TF-1 cells were left untreated (control) or
were treated with 5 mmol/L Na-Sal for 5 hours with or without 1-hour
pretreatment with 100 µmol/L Z-VAD. fmk (Calbiochem) as indicated.
(B) z-VAD prevents caspase-3 activation. Cells were left untreated
(control) or were treated with 5 mmol/L Na-Sal alone or in the presence
of 100 µmol/L Z-VAD for 16 hours. Caspase-3 activity was determined
as described in Materials and Methods. (C and D) Z-VAD prevents
Na-Sal-induced cleavage of gelsolin (C) and PARP (D). Cells were
untreated (lane 1), or treated with 5 mmol/L Na-Sal for 5 hours in the
absence (lane 2) or presence of 100 µmol/L Z-VAD (lane 3). The assays
for PARP and gelsolin cleavage are described in the legend to Fig 3.
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Na-Sal potentiates apoptosis induced by growth factor withdrawal or
daunorubicin.
The effectiveness of cancer chemotherapy is determined by its ability
to trigger apoptosis in malignant cells and its toxicity profile. We
investigated whether Na-Sal could sensitize TF-1 cells to apoptosis
induced by daunorubicin, a chemotherapeutic agent commonly used in the
treatment of human acute myeloid leukemias, or with growth factor
withdrawal-induced apoptosis.
Although TF-1 cells are dependent on GM-CSF for growth, there was no
significant effect of GM-CSF withdrawal on the viability of TF-1 cells
within the first 24 hours (Fig 5A).
Treatment of TF-1 cells with 3 mmol/L Na-Sal in the presence of GM-CSF
decreased the growth rate of TF-1 cells slightly after 24 hours of
treatment, whereas treatment of TF-1 cells with 3 mmol/L Na-Sal in the
absence of GM-CSF triggered rapid apoptosis, resulting in complete cell death within 24 hours. This result suggests that at low concentrations, Na-Sal can potentiate apoptosis triggered by growth factor deprivation.
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| Fig 5.
Na-Sal enhances growth factor withdrawal-induced and
daunorubicin-induced apoptosis of TF-1 cells. (A) TF-1 cells were grown
in the presence or absence of GM-CSF and treated with 3 mmol/L Na-Sal.
Viability was determined by trypan blue dye exclusion at the times
indicated. (B) TF-1 cells were treated with daunorubicin (Dauno) alone
(0.1 µg/mL or 0.5 µg/mL), Na-Sal alone (2 mmol/L) or the
combination of daunorubicin (0.1 µg/mL) and 2 mmol/L Na-Sal for the
times indicated. Viability was determined using the MTT assay. These
experiments were repeated three times with similar results. Results of
a representative experiment are shown.
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To determine whether Na-Sal also sensitizes TF-1 cells to undergo
daunorubicin-induced apoptosis, we compared the rate of apoptosis in
cells treated with sublethal concentrations of daunorubicin (0.1 µg/mL) or Na-Sal (2 mmol/L) with the rate of apoptosis in cells
treated with the combination of both agents. There was little effect on
the viability of TF-1 cells after 48 hours exposure to either agent
alone, however, in the presence of both 2 mmol/L Na-Sal and 0.1 µg/mL
daunorubicin, TF-1 cells underwent significant cell death as early as 3 hours after treatment. In fact, these cells died as rapidly as cells
treated with 0.5 µg/mL of daunorubicin (Fig 5B). This result shows
that Na-Sal and daunorubicin can synergize to induce apoptosis in TF-1 cells.
| |
DISCUSSION |
In this study, we report that several myeloid leukemia cell lines
(TF-1, CMK-1, U937, HL-60, Mo7e) undergo rapid apoptosis on incubation
with concentrations of salicylates comparable to those achieved in the
plasma of patients treated for inflammatory disorders (1 to 3 mmol/L).37-39 The reduced sensitivity of K562 cells to
Na-Sal-induced apoptosis may be due to the presence of the t(9;22)
chromosomal translocation and the expression of the bcr-abl fusion
protein in this cell line. Bcr-abl has been shown to suppress growth
factor withdrawal-induced apoptosis in BaF3 cells in a BCL-2-dependent
manner40 and may protect cells from Na-Sal-induced
apoptosis, as well. Na-Sal is a poor inhibitor of COX1 and COX2, thus
inhibition of prostaglandin synthesis most likely does not play a
significant role in Na-Sal-induced apoptosis. Our data also show that
Na-Sal can trigger apoptosis independent of p53, because TF-1 cells (as
well as U937, HL-60, and CMK-1 cells) lack functional p53 protein.
We have shown that Na-Sal affects both the effector and the execution
phases of apoptosis. The response of cells to apoptotic signals
critically depends on the balance between the proapoptotic and
antiapoptotic members of the BCL-2 family.26 While Na-Sal did not affect the expression of BCL-2, BCL-XL, or Bax
proteins in TF-1 cells, it inhibited MCL-1 expression in a time- and
dose-dependent manner. MCL-1 is an antiapoptotic member of the BCL-2
family41 and we and others have previously shown that
downregulation of MCL-1 expression precedes growth factor
withdrawal-induced apoptosis of TF-1 cells.24 Thus,
inhibition of MCL-1 protein expression may also play a role in
mediating Na-Sal-induced apoptosis. MCL-1 is strongly upregulated by
GM-CSF in TF-1 cells23 and we showed that GM-CSF delays
Na-Sal-induced apoptosis, further supporting the role of MCL-1 in
Na-Sal-induced apoptosis. We have also shown that Na-Sal treatment
results in decreased levels of MCL-1 mRNA (data not shown), suggesting
that salicylates inhibit expression of MCL-1 at the transcriptional
rather than the translational level. Although NF-B activity is
strongly inhibited by salicylates,16 the downregulation of
MCL-1 expression by Na-Sal is unlikely to be mediated directly by
inhibition of NF-B activity because the MCL-1 promoter apparently
lacks a potential NF-B binding site (R. Craig, personal
communication, June 1998).
The execution phase of programmed cell death involves the activation of
caspases and subsequent cleavage of several cellular substrates such as
PARP, gelsolin, actin, lamins, and fodrin.33-36 Caspase-3
is a likely candidate to mediate Na-Sal-induced apoptosis, as we
showed both the proteolytic activation of the enzyme, as well as the
increase in protease activity of caspase-3 in Na-Sal-treated cells
(Fig 3A and B). However, we cannot exclude the involvement of other
caspases in Na-Sal-induced cell death. We have found that caspase-7, a
close relative of caspase-3, is highly expressed in TF-1 cells, and we
have observed the disappearance of the proenzyme form of caspase-7 in
Na-Sal-treated cells (data not shown). It is thus probable that
caspase-7 also plays a role in Na-Sal-induced apoptosis. In contrast,
caspase-9, a caspase located upstream of caspase-3 in the caspase
signaling cascade42 is not expressed in TF-1 cells, thus
other CARD (for caspase recruitment domain) containing caspases (such
as caspases 1-, 2-, and 8) may activate caspase-3 in this system.
The increased caspase-3 activity in Na-Sal-treated cells is
accompanied by cleavage of PARP and gelsolin. PARP is a 116-kD protein,
which converts nicotinamide adenine dinucleotide (NAD) to nicotinamide
and protein-linked ADP-ribose polymers. In response to growth factor
withdrawal or on exposure to a variety of chemotherapeutic compounds,43 PARP is cleaved to generate an 85-kD fragment. Whereas cleavage of PARP is typically observed during apoptosis, recent
findings indicate that its cleavage may not be necessary for the
process of apoptosis.44 Cleavage of gelsolin, however, may
be essential for apoptosis, because the gelsolin cleavage product
itself is sufficient to cause the morphological changes accompanying
apoptosis, such as cell rounding, blebbing, and nuclear fragmentation.36 Moreover, downregulation of gelsolin
expression is seen in many cancer cells,45,46 potentially
contributing to the resistance of tumor cells to apoptosis. The caspase
inhibitor, Z-VAD, prevented both Na-Sal-induced cleavage of PARP and
gelsolin and apoptosis of TF-1 cells, establishing an essential role
for caspase activation in Na-Sal-induced apoptosis.
The resistance of cancer cells to chemotherapy-induced apoptosis
remains one of the most significant problems in the treatment of
cancer.47 Resistance may occur due to mutations of p53,
overexpression of BCL-2, BCL-XL, or MCL-1 or downregulation
or mutations of BAX.48-50 Cells that overexpress MCL-1 are
highly resistant to chemotherapeutic agents in vitro,51 and
MCL-1 levels were recently shown to be elevated in acute myeloid
leukemia cells at the time of leukemic relapse,52 when they
are often resistant to chemotherapy. Likewise, inhibition of MCL-1
expression by transfection with antisense-MCL-1 c-DNA decreased the
viability of TF-1 cells,23 and treatment of animals with
antisense oligonucleotides that inhibit the expression of BCL-2 has
been shown to increase the effectiveness of chemotherapy.53 Thus, our finding that Na-Sal can efficiently inhibit the expression of
MCL-1 holds promise for improving the treatment of leukemia (and
possibly other malignancies, as well).
Our data show that salicylates accelerate growth factor
withdrawal-induced apoptosis and synergize with sublethal
concentrations of daunorubicin to induce programmed cell death.
Potential mechanisms that could account for this synergism are
currently being explored in the laboratory. Daunorubicin stimulates
ceramide synthase in hematopoietic cells and induces apoptosis via
generation of ceramide,54 which, like Na-Sal, induces
caspase-3 activation and cleavage of PARP.55 This suggests
that cellular targets other than caspase-3 most likely mediate the
synergism between daunorubicin and Na-Sal.
Daunorubicin is also a potent inducer of NF-B, which has been shown
to lower its killing efficiency.19,56 Thus, inhibition of
daunorubicin-induced NF-B activation by Na-Sal could account for the
more efficient killing that occurs after exposure to both agents.
Indeed, inhibition of NF-B has been recently shown to significantly
enhance drug- or death ligand-induced apoptosis in lymphoid cell lines,
as well as in primary leukemia cells from patients with acute
lymphoblastic leukemia (ALL) and acute myelogenous leukemia
(AML).57 Daunorubicin does not modulate
the expression of MCL-1 in ML-1 cells58 or TF-1 cells (data
not shown), thus downregulation of MCL-1 by Na-Sal could be important
for its ability to enhance killing by daunorubicin. Moreover,
DNA-damaging agents, such as ionizing radiation, UV radiation, and
alkylating drugs can increase MCL-1 expression in human myeloid
leukemia cells,59 and it will be important to test whether
Na-Sal can affect killing induced by these agents. While this report
was being submitted, Bellosillo et al60 reported that
aspirin and salicylate induce apoptosis of primary B-cell chronic
lymphocytic leukemia (B-CLL) cells in concentrations comparable to the
ones used in our study and that mononuclear cells from normal donors
are significantly more resistant to aspirin-induced apoptosis. Our
preliminary studies, using CD34+ cells isolated from
umbilical cord blood, show that concentrations of Na-Sal that inhibit
the growth of many myeloid leukemia cell lines, do not inhibit the in
vitro growth of burst-forming unit-erythroid (BFU-E)
or colony-forming unit-granulocyte-macrophage (CFU-GM) (data not
shown); higher concentrations of Na-Sal (10 mmol/L) do inhibit colony
formation in vitro.
Hence, our data raise the possibility that Na-Sal could be used to
lower the apoptotic threshold of leukemia cells and potentially improve
the treatment of human myeloid leukemia.
| |
ACKNOWLEDGMENT |
We thank Drs Inge Olsson and Donal MacGrogan for comments and critical
reading of the manuscript.
| |
FOOTNOTES |
Submitted September 4, 1998; accepted November 30, 1998.
Supported by the DeWitt Wallace Research Fund, the Sunshine Lady
Foundation, the Samuel P. Reed Fund, and by United States Public Health
Service Grants No. DK43025 and DK52208.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Stephen Nimer, MD, Department of Medicine,
Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY
10021; e-mail: s-nimer{at}ski.mskcc.org.
| |
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TOP
ABSTRACT
INTRODUCTION